High mobility of flap endonuclease 1 and DNA polymerase eta associated with replication foci in mammalian S-phase nucleus.
ABSTRACT Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase eta (Pol eta). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Pol eta and GFP-Fen1 are mobile with residence times at RFi (t(m)) approximately 2 and approximately 0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and t(m) increased, but it then recovered. The mobilities of focal GFP-Pol eta and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks.
- SourceAvailable from: pnas.org[show abstract] [hide abstract]
ABSTRACT: The cause of increased radiosensitivity in ataxia-telangiectasia (AT) cells may be a defect in their ability to respond to DNA damage rather than a defect in their ability to repair it. Doses of x-radiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition in AT cells and thus less delay during which x-ray damage could be repaired. The radioresistance of DNA synthesis in AT cells was primarily due to a much smaller inhibition of replicon initiation than in normal cells; the AT cells were also more resistant to damage that inhibited chain elongation. AT cells have been reported to undergo less radiation-induced mitotic delay than normal cells, which may cause them to move from G2 phase into mitosis before repair is complete and may result in the increased incidence of chromatid aberrations observed by others. Therefore, AT cells fail to go through those delays that allow normal cells to repair DNA damage before it can be expressed.Proceedings of the National Academy of Sciences 01/1981; 77(12):7315-7. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Treatment of normal human fibroblasts (NHFs) with cycloheximide, which inhibits protein synthesis, resulted in partial inhibition of their DNA synthesis, as determined by incorporation of radioactive thymidine and resistance of the cells to subsequent treatment with bleomycin. The effects of treatments of ataxia telangiectasia fibroblasts (ATFs) with cycloheximide and then bleomycin on their DNA synthesis were very similar to those on DNA synthesis of NHFs. The fact that treatment with bleomycin only caused transient inhibition of DNA synthesis within an hour in NHFs but not ATFs was confirmed. Studies by alkali-density gradient centrifugation showed that the cycloheximide mainly inhibited formation of short fragments of DNA in both NHFs and ATFs, as bleomycin does in NHFs. These findings suggest that these two chemicals both inhibit replicon initiation, and thus provide evidence that the genetic defect in ATFs is related to replicon initiation.Cell Biology International Reports 12/1988; 12(11):943-50.
- [show abstract] [hide abstract]
ABSTRACT: We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1's DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.Molecular Cell 07/2001; 7(6):1221-31. · 15.28 Impact Factor
Molecular Biology of the Cell
Vol. 16, 2518–2528, May 2005
High Mobility of Flap Endonuclease 1 and DNA
Polymerase ? Associated with Replication Foci in
Mammalian S-Phase Nucleus□
Lioudmila Solovjeva,* Maria Svetlova,* Lioudmila Sasina,†Kyoji Tanaka,‡
Masafumi Saijo,‡Igor Nazarov,§Morton Bradbury,§?and Nikolai Tomilin*
*Laboratory of Chromosome Stability, Institute of Cytology, Russian Academy of Sciences, 194064 St.
Petersburg, Russia;†Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St.
Petersburg, Russia;‡Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan;
§Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis,
CA 95616; and?Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545
Submitted December 10, 2004; Revised February 15, 2005; Accepted February 21, 2005
Monitoring Editor: Joseph Gall
Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green
fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using
fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the
existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged
PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase ? (Pol?). We also used the GFP-Cockayne
syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells,
GFP-Pol? and GFP-Fen1 are mobile with residence times at RFi (tm) ?2 and ?0.8 s, respectively. GFP-CSA is also mobile
but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal
GFP-Fen1 decreased and tmincreased, but it then recovered. The mobilities of focal GFP-Pol? and GFP-PCNA did not
change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal
replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after
DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks.
In mammalian cells, DNA replication during S phase is
located at distinct nuclear sites. Replication foci (RFi) each
contain on average 10 spatially clustered active replication
forks that can be visualized using antibodies against incor-
porated halogenated deoxyuridines (Nakamura et al., 1986;
Nakayasu and Berezney, 1989; van Dierendonck et al., 1989;
O’Keefe et al., 1992; Tomilin et al., 1995; Jackson and Pombo,
1998). In fixed cells, the RFi colocalize with the Triton-
insoluble form of the replication protein proliferating cell
nuclear antigen (PCNA) (Bravo and McDonald-Bravo, 1987),
the p70 subunit of replication protein A (RPA70), protein
kinase Cdk2 and cyclin A (Cardoso et al., 1993), DNA poly-
merase ? (Hozak et al., 1993), DNA ligase I (Cardoso et al.,
1997), and DNA polymerase ? (Pol?) (Kannouche et al.,
2001). Distinct nuclear sites accumulate green fluorescent
protein (GFP)-tagged DNA ligase I (Cardoso et al., 1997),
GFP-PCNA (Leonhardt et al., 2000; Somanathan et al., 2001).
The foci containing GFP-RPA34 (Sporbert et al., 2002) or
GFP-Pol? (Kannouche et al., 2001) also can be observed in
normally proliferating living S-phase cells, indicating that
the RFi seen in fixed cells do not arise during the fixation
procedure but reflect real clustering of replication forks and
associated molecular machines in the nucleus.
It was initially suggested that the RFi represent special-
ized subnuclear organelles or nucleoskeleton-attached rep-
lication factories in which preassembled holocomplexes
(replisomes) duplicate incoming DNA (Hozak et al., 1993). In
agreement with this model, recent studies of living cells
expressing GFP-tagged PCNA by using the fluorescence
redistribution after photobleaching (FRAP) assay (Axelrod
et al., 1976; Lippincott-Schwartz et al., 2001) showed that
focal GFP-PCNA exchanged very slowly (Sporbert et al.,
2002). During S phase, immobile GFP-PCNA foci can assem-
ble and disassemble in the nucleus with lifetimes ranging
from 30 min to 3 h (Leonhardt et al., 2000), consistent with
significant heterogeneity of replication units and the exis-
tence of very large replicons (Liapunova, 1994). However,
another replication protein, GFP-RPA34, was found to be
mobile at RFi and after bleaching completely redistributed
within 2 min (Sporbert et al., 2002). This indicates that de-
spite their existence as morphologically distinct nuclear bod-
ies, replication factories may contain mobile components.
Transcription factories (Jackson et al., 1993) and nucleotide
excision repair foci (Jackson et al., 1994; Svetlova et al., 2002)
have been visualized in mammalian cells, and recent studies
indicate that they are stochastically assembled de novo in
each round of transcription or repair from freely diffusible
components (Houtsmuller et al., 1999; Dundr et al., 2002;
This article was published online ahead of print in MBC in Press
on March 9, 2005.
DThe online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Nikolai Tomilin (email@example.com).
2518 © 2005 by The American Society for Cell Biology
Kimura et al., 2002). Cockayne syndrome group A (CSA)
protein is required for the excision of DNA lesions at stalled
transcription sites, also called transcription-coupled repair
(TCR) (Hanawalt, 2002). After UV-irradiation, a fraction of
this protein becomes insoluble in a buffer containing Triton
X-100, and this insoluble CSA shows colocalization with
active (phosphorylated) RNA polymerase II foci (Kamiuchi
et al., 2002). However, possible changes of mobility of CSA
protein after DNA damage have not been studied so far.
It is known that strand elongation during DNA replication
can be inhibited by DNA lesions induced by UV-irradiation
and some chemical carcinogens, e.g., methyl methanesulfo-
nate (MMS) (Merrick et al., 2004), and this leads to the
accumulation of S-phase cells with stalled replication forks
(Kannouche et al., 2001, 2004). In these cells, clusters of
blocked replication forks colocalize with stably bound
PCNA and Pol?, which is required for translesion DNA
synthesis across damaged sites (Masutani et al., 1999). PCNA
and Pol? associated with normal or stalled replication foci
are insoluble in methanol or in a buffer containing Triton
X-100 (Celis and Madsen, 1986; Bravo and McDonald-Bravo,
1987; Toschi and Bravo, 1988; Kannouche et al., 2001).
In yeast, Pol? (Rad30p)-dependent translesion synthesis
requires the monoubiquitination of PCNA performed by the
Rad6p/Rad18p complex (Stelter and Ulrich, 2003) and the
Rad6 epistasis group (controlling lesion bypass), which in
addition to the Rad30 and Rad18 genes also contains the
Rad27 gene encoding the structure-specific Flap nuclease
Fen1 (Reagan et al., 1995). The yeast Fen1 protein, which is
involved in the maturation of Okazaki fragments, sup-
presses the accumulation of single-stranded DNA (ssDNA)
and double-strand breaks (DSBs) at replication forks (De-
brauwere et al., 2001). Mammalian Rad18 protein and Pol?
are required for lesion bypass (Cordeiro-Stone et al., 1997;
Masutani et al., 1999; Tateishi et al., 2000; Limoli et al., 2002;
Tateishi et al., 2003); mammalian Rad18 also monoubiquiti-
nates Pol? at stalled replication forks (Kannouche et al., 2004;
Watanabe et al., 2004). How DNA damage induces activation
of the Rad18 protein is unknown, but some data suggest that
the ATR-Chk1 checkpoint pathway may be involved (Niki-
forov et al., 2004). The Fen1 (?/?) mouse is not viable
(Kucherlapati et al., 2002), and expression of nuclease-defec-
tive Fen1 inhibits S-phase progression after MMS treatment
(Shibata and Nakamura, 2002), suggesting that Fen1 may be
required for efficient lesion bypass.
Here, we used the FRAP assay to study the mobilities of
GFP-tagged human proteins Fen1, Pol?, PCNA, and CSA in
transfected Chinese hamster cells before and after the induc-
tion of DNA damage by MMS or UV. We found that in
normally proliferating cells focal GFP-Fen1 and GFP-Pol?
are very mobile, supporting the concept of self-organization
of cellular architecture (Misteli, 2001).
MATERIALS AND METHODS
Cells, Transfections, and Treatments
Chinese hamster V79-4 immortal lung fibroblasts and human A539 cells were
obtained from American Type Culture Collection (Manassas, VA). Cells were
cultivated in RPMI 1640 medium supplemented with 10% of fetal calf serum
(FCS). Transient transfections with the indicated plasmids involved the
TRANS-FAST reagent (Promega, Madison, WI), and cells were analyzed
24–48 h after transfection. Stable expressing clones were isolated after tran-
sient transfection by selection in growth medium containing 0.6–1 mg/ml
G418 (Geneticin). For isolation of a stable clone expressing GFP-Fen1, the
corresponding plasmid was introduced into V79-4 cells. A plasmid encoding
GFP-CSA protein was introduced into CS3BESV cells (SV40-transformed cells
from a patient with Cockayne syndrome group A, cultivated in DMEM with
10% FCS), and UV-sensitivity of the obtained clone (OS-7) was compared with
the UV-sensitivity of the normal fibroblasts line WI38VA13 as described
previously (Kamiuchi et al., 2002). Expressing clones were identified by GFP
fluorescence and then expanded. MMS (Aldrich Chemical, Milwaukee, WI)
was added to cells in growth medium to the final concentration of 0.01 or
0.03% for 1 h. Then, cells were washed with phosphate-buffered saline (PBS)
and incubated in growth medium for the time intervals indicated.
Sources of Plasmids Encoding GFP-tagged Proteins
Plasmids encoding GFP-Fen1 and GFP-PCNA were constructed for this study
as described below. DNA fragments containing the cDNAs of human Fen1
and PCNA were amplified from human polyA-containing RNA by using the
single-tube Titan reverse transcription-PCR kit (Roche Diagnostics, Indianap-
olis, IN). The PolyA-containing RNAs were isolated from cultivated human
A539 cells by using the mRNA isolation kit (Roche Diagnostics). The se-
quences of primers used to amplify human Fen1 cDNA were Fen-C1 CTGT-
GTTGCCATGGGAATTC and Fen-C2 TTCCCCTTTTAAACTTCCCTGC. The
sequences of primers to amplify human PCNA cDNA were PCNA-C1
GATCTTGGG. For the construction of expression plasmids GFP-Fen1 and
GFP-PCNA we used C-terminal fusion GFP-TOPO vectors from Invitrogen
(Carlsbad, CA) and chemically competent TOP10 cells. Individual clones
were screened by PCR, and the presence of the expected inserts was con-
firmed by sequencing. Plasmid DNAs were purified using a QIAGEN kit
(QIAGEN, Valencia, CA). Analysis of Chinese hamster cells transfected with
the GFP-Fen1 plasmid involved Western blotting with anti-GFP antibodies
(Zymed Laboratories, South San Francisco, CA) and showed presence of the
fusion protein of expected size (?70 kDa). The plasmid encoding GFP-tagged
DNA polymerase ? (eGFP-Pol?) was obtained from A. R. Lehmann (Univer-
sity of Sussex, Brighton, United Kingdom). The plasmid encoding GFP-CSA
was constructed by subcloning of the PCR-amplified CSA coding sequence
(Kamiuchi et al., 2002) into the vector pEGFP-N3 (BD Biosciences Clontech,
Palo Alto, CA).
Immunofluorescence Detection of PCNA and RNA
Polymerase II in Fixed Cells
GFP-Fen1–expressing cells grown on glass slides were washed with PBS,
extracted overnight at –20°C in 100% methanol, and then fixed in 4% form-
aldehyde. PCNA was visualized using mouse monoclonal antibodies PC-10
(1:100, 30 min; LabVision/NeoMarkers, Fremont, CA) and anti-mouse IgG
coupled to Alexa Fluor 568 (1:400, 30 min; Molecular Probes, Eugene, OR). For
the detection of PCNA and RNA polymerase II, GFP-Pol? and GFP-CSA–
expressing cells were washed in PBS and then extracted (Kamiuchi et al., 2002)
for 20 min at room temperature in cytoskeleton (CSK) buffer containing 10
mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM
dithiothreitol (DTT), mM EGTA, and 0.5% Triton X-100 and then washed in
PBS and fixed in 4% formaldehyde. PCNA was then visualized in GFP-Pol?–
expressing cells by using mouse monoclonal antibodies PC-10 (1:100, 30 min;
Santa Cruz Biotechnology, Santa Cruz, CA), biotinylated sheep anti-mouse
IgG (1:100, 30 min; Sigma-Aldrich, St. Louis, MO), and avidin-Texas Red
(1:200, 30 min; Vector Laboratories, Burlingame, CA). RNA polymerase II in
GFP-CSA–expressing cells was detected using mouse monoclonal antibodies
against phosphorylated RNAP II (clone H5) obtained from BAbCO (Rich-
mond, CA) (1:200, 60 min) followed by the secondary anti-mouse antibodies
coupled to Alexa Fluor 568.
Immunoprecipitation and Immunoblotting
For coimmunoprecipitation (CIP), cell lysates were prepared using radioim-
munoprecipitation assay (RIPA) buffer (1 ml/100-mm plate) to which (after
clearing lysates by centrifugation) 5 ?l of undiluted antibodies was added.
After incubation for 1 h on ice, immune complexes were isolated using
protein A coupled to Sepharose beads (100 ?l of 10% suspension per 1 ml of
initial lysate), which before SDS electrophoresis, were washed five times with
RIPA buffer. Then, pellets were suspended in 2? Laemmli buffer (2% SDS,
10% glycerol, 100 mM DTT, 60 mM Tris-HCl, pH 6.8, and 0.001% bromphenol
blue) and heated at 85°C for 10 min. RIPA buffer contained 50 mM Tris-HCl
buffer, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecylsulfate,
and 0.5% sodium deoxycholate. For CIP, we used mouse monoclonal anti-
bodies (clone 14A1) against human recombinant Fen1 (catalog no. MS-1752;
NeoMarkers), mouse monoclonal antibodies (clone JL-8) against GFP (catalog
no. 8371-2; BD Biosciences Clontech), and custom-made rabbit polyclonal
CLTFGS(PO4)PVLMRHLTA-C? (Genemed Synthesis, South San Francisco,
CA). As negative CIP controls, affinity-purified polyclonal antibodies against
CSB or CSA proteins (sc-10459 and sc-10997, resp.; Santa Cruz Biotechnology)
or preimmune rabbit serum (Genemed Synthesis) was used. On immuno-
blots, coimmunoprecipitated PCNA was detected using mouse monoclonal
antibodies PC-10 (catalog no. MS-106-P1; NeoMarkers). After separation of
proteins in denaturing SDS polyacrylamide gels, they were transferred to a
membrane and then processed as described previously (Siino et al., 2002;
Nazarov et al., 2003). For chemiluminescence detection of Fen1 and GFP-
tagged protein, we used the same primary monoclonal mouse monoclonal
antibodies described above.
Protein Mobility at Replication Foci
Vol. 16, May 20052519
The FRAP assay (Axelrod et al., 1976; Lippincott-Schwartz et al., 2001) in-
volved the photobleaching of a small area in the nucleus at maximal argon
laser power followed by serial scans at a lower magnification and laser power.
All FRAP assays were carried out on cells in four-well Lab-Tek (Naperville,
IL) coverglass chambers. Original FRAP curves of relative fluorescence (RF)
intensity were calculated as RF(t) ? It/Ipre, where Ipreis the prebleach value
for each measurement, and Itis the fluorescence at the time point t. After
correction for the nonspecific fluorescence loss during the image acquisition,
the data obtained under identical conditions for at least 10 cells for each
variant were averaged, and the SE was calculated for each time point. To
determine the representative residence time (tm) of a protein, the relative
fluorescence was calculated and further normalized from the original aver-
aged FRAP curves as RFrt(t) ? (It– I0)/(Ipost– I0), where I0is the relative
fluorescence intensity immediately after bleaching, and Ipostis the maximal
relative fluorescence intensity observed after recovery (taken as 1). Average
residence time (tm) for a given protein was then estimated as the time
required for recovery of 63.2% of the final fluorescence (Lukas et al., 2004). To
determine mobile fractions the relative fluorescence was calculated and fur-
ther normalized from the original averaged FRAP curves as RFmf(t) ?
(It– I0)/(Ipre– I0), where Ipreis the relative fluorescence before bleaching (for
normalized sets taken as 1), and I0is the fluorescence intensity just after
bleaching. The mobile fraction is then determined as maximal value of RFmf
after recovery. In a Zeiss LSM 510, the first scan at low laser intensity after
bleaching takes at least 100 ms, but it is tentatively taken throughout this
article that the fluorescence intensity at the end of the first scan at low laser
intensity is zero point, because the actual zero point fluorescence immediately
after bleaching is impossible to measure.
Images of live and fixed cells and FRAP assay were obtained with a Zeiss
confocal laser scanning system LSM-510 equipped with a Plan-NEOFLUAR
63/1.3 objective, helium-neon laser of 543 nm wavelength, and/or argon laser
(15 mW) of 458/488-nm wavelengths, or a MRC-1024 fluorescence micros-
copy system (Bio-Rad).
Expression of GFP-tagged Replication/Repair Proteins in
In Chinese hamster cells transiently transfected with the
fusion plasmid encoding Fen1-C-GFP, two distinct distribu-
tions of the expressed GFP in nuclei were found: one show-
ing only diffuse nuclear fluorescence and the other showing
both diffuse and focal GFP nuclear signal (Figure 1A). Sim-
ilar patterns of GFP labeling were shown in cells from a
stable GFP-Fen1–expressing clone which was selected in the
growth medium containing G418. Analysis of these cells by
Western blotting by using antibodies against GFP showed
the presence of a single band of protein of the size expected
for the fusion protein (Figure 1B, left) with sensitivity to
MMS very similar to that of the parental V79 cells (?15%
survival after 0.01% MMS), indicating that the GFP-tagged
Fen1 does not interfere with the repair functions of endog-
enous Fen1. In stably transformed cells the fusion GFP-Fen1
protein is expressed at about the same level as the endoge-
nous Fen1 (Figure 1B, right blot).
To show that the GFP-Fen1 fusion protein stably ex-
pressed in Chinese hamster cells retains its function, we
used CIP assay to determine whether it interacts with
PCNA. For CIP, we used mouse monoclonal antibodies
against human recombinant Fen1 (Figure 2A, lane 2), mouse
monoclonal antibodies against GFP (Figure 2A, lane 3), and
rabbit polyclonal antibodies against Fen1 peptide (Figure
2A, lane 5). As controls, affinity-purified polyclonal antibod-
ies against CSB or CSA proteins (lanes 2 and 4, respectively)
or preimmune rabbit serum (lane 6) were used. It is seen
from Figure 2A that PCNA can be coprecipitated from ly-
sates of GFP-Fen1–expressing cells with antibodies against
Fen1 (Figure 2A, lanes 2 and 5) as well as with antibodies
against GFP (lane 3) but not with antibodies against CSB
protein (lane 2), CSA protein (lane 4) or with preimmune
rabbit serum (lane 6). PCNA also has been shown to immu-
noprecipitate by anti-Fen1 and anti-GFP antibodies with
equal efficiencies (Figure 2B). These results confirm that
Fen1 protein fused at its C terminus to GFP interacts with
To confirm that GFP-Fen1 concentrates at replication foci,
we examined its colocalization with PCNA foci after preex-
traction of cells with 100% methanol, which removes soluble
PCNA, leaving only molecules involved in DNA replication
(Madsen and Celis, 1985), or with CSK buffer containing
0.5% Triton X-100 (Kamiuchi et al., 2002). We found that the
focal GFP-Fen1 in transiently transfected cells preextracted
with CSK buffer colocalized with PCNA (Figure 2B and
Supplementary Figure 1A). Similar colocalization of the fo-
cal GFP signal and PCNA was observed in preextracted V79
cells transiently transfected with the plasmid expressing
GFP-tagged Pol? (Figure 2C and Supplementary Figure 1B),
confirming previous observations (Kannouche et al., 2001).
The plasmid expressing GFP-Pol? used in our study has
been shown to be able to complement a repair defect in XP
variant cells (Kannouche et al., 2001) and therefore encodes
a fully functional fusion protein. Pol?, which has a con-
served domain for PCNA binding, is known to interact with
PCNA in undamaged cells (Haracska et al., 2001). We also
confirmed colocalization of GFP-Fen1 and PCNA in S-phase
cells of a stable clone preextracted with methanol (Supple-
mentary Figure 1C). Because PCNA is well known to be the
main factor in recruiting Fen1 to replication forks (Li et al.,
1995; Jonsson et al., 1998; Gomes and Burgers, 2000; Tom et
al., 2000), our results indicate that the fusion of the C termi-
nus of Fen1 with the N terminus of GFP obtained in this
Chinese hamster V79 cells. (A) Two patterns of GFP-Fen1 distribu-
tion in the nuclei of transiently transfected live cells: diffuse (left)
and focal (right). Bar, 10 ?m. (B) Western blots of total protein from
stably transfected V79 cells probed with antibodies against human
recombinant Fen1 (right) or GFP (left). Endogenous Fen1 protein is
marked as e-Fen1.
Expression of GFP-tagged human Fen1 in transfected
L. Solovjeva et al.
Molecular Biology of the Cell2520
study does not effect the Fen1 nuclear localization signal
(Qiu et al., 2001) nor its binding to PCNA during replication.
To study the function of the GFP-CSA protein, its plasmid
was transfected into human UV-sensitive cells deficient in
endogenous CSA protein, resulting in a stable expression
clone OS-7 (Figure 3A, left two images) that was found to be
UV-resistant (Figure 3B). The expressed fusion protein of
expected size was detected in this clone by anti-GFP anti-
bodies (Figure 3C). The CSA protein does not interact with
PCNA, and GFP-CSA can be almost completely extracted
from unirradiated OS-7 cells with a cytoskeleton buffer con-
taining 0.5% Triton X-100 (Figure 3A, right top image). How-
ever, GFP-CSA becomes resistant to extraction with the in-
dicated buffer after UV-irradiation (Figure 3A, right bottom
image), confirming previous observations (Kamiuchi et al.,
2002). In many preextracted UV-irradiated cells, Triton-in-
soluble GFP-CSA partially colocalizes with active RNA
polymerase II (Supplementary Figure 2). Interestingly, the
treatment of cells with an inhibitor of histone deacetylases
trichostatin A (but not with an inhibitor of protein kinases
roscovitine) leads to a significantly increased amount of
Triton-insoluble GFP-CSA in UV-irradiated cells (Figure
3D). This drug does not induce a significant increase of
overall expression of GFP-CSA in OS7 cells (our unpub-
lished data), suggesting that the UV-induced insolubiliza-
tion of CSA protein (Kamiuchi et al., 2002) may involve
interactions of this protein with chromatin containing hy-
Our GFP-PCNA construct in which GFP is fused to the
PCNA C terminus is also likely to be functional because it
associates with replication foci and becomes diffusionally
immobile (see below), confirming previous observations
(Sporbert et al., 2002). It also may be noted that in another
study, the C-fusion of PCNA to GFP showed the expected
behavior at replication foci (Somanathan et al., 2001), sug-
gesting that GFP linked to C-end of PCNA does not interfere
Focal GFP-Fen1 and GFP-Pol? Are Mobile in Normally
Proliferating Living Cells
The FRAP analyses of the mobilities of GFP-Fen1 and GFP-
Pol? are shown in Figure 4. It is seen that fluorescence of the
focal GFP-Fen1 in transiently (Figure 4, A and B) or stably
expressing (Figure 4, C and D) undamaged Chinese hamster
cells is recovered after photobleaching after a few seconds. A
similar rapid recovery in transiently transfected V79 cells
was found for focal GFP-Pol? (Figure 4, E and F), indicating
that both these PCNA-interacting proteins dynamically in-
teract with the replication machinery in S-phase cells and are
in continuous exchange with the pool of nucleoplasmic pro-
tein during DNA synthesis. Using the FRAP assay, we also
analyzed the mobilities of diffuse nuclear GFP-Fen1, GFP-
Pol?, and diffuse and focal GFP-PCNA and calculated the
average redistribution times of these proteins (Table 1) and
their mobile fractions (Table 2) as described in Materials and
Methods. It is seen from Table 1 that in a stably expressing
clone, diffuse GFP-Fen1 is redistributed in 0.38 s, whereas
focal GFP-Fen1 is redistributed in 0.78 s, thereby explaining
the observation of distinct GFP-Fen1 foci in living cells. A
similar increase in the redistribution times of the focal com-
ponent (2.05 s) compared with the diffuse component (1.01 s)
is observed in cells transiently transfected with GFP-Pol?
(Table 1). The mobile fractions of focal GFP-Fen1 and GFP-
Pol? in undamaged cells are found to be 0.89 and 1.03,
respectively (Table 2, first row), indicating that major frac-
tions of these proteins are mobile in the nucleus. The redis-
tribution times of the focal components are equivalent to
their representative residence times in foci (Lukas et al.,
2004), so GFP-Fen1 associates with replication foci for ?1 s
and GFP-Pol? for ?2 s. Diffuse GFP-PCNA also is mobile
and showed a redistribution time of 0.66 s, but the focal
GFP-PCNA is very slowly exchanged (Table 1), confirming
the observations of other authors (Sporbert et al., 2002).
The GFP-CSA protein, which does not interact with
PCNA, showed only a diffuse component in the nucleus
(Figure 3). The FRAP analysis of its mobility in transiently or
stably expressing cells (Figure 5, A and B) indicates that this
protein also is mobile, with a redistribution time in stably
expressing OS-7 cells of ?4 s (Table 1). This tmvalue is ?10
times higher than that for diffuse GFP-Fen1, although these
proteins have approximately the same size (70 kDa), which
enous PCNA in Chinese hamster cells. (A) Immunoprecipitation of
endogenous PCNA from cell lysates of V79 cells stably expressing
GFP-Fen1 with antibodies against human recombinant Fen1 (hr-
Fen1, lane 1), GFP (lane 3), or Fen1 peptide (lane 5). As controls,
affinity-purified antibodies against CSB protein (lane 2), CSA pro-
tein (lane 4) or preimmune serum (lane 6) were used. (B) Compar-
ative PCNA immunoprecipitation with antibodies against GFP (lane
2) and Fen1 peptide (lane 3). Lane 1 shows input. All blots were
probed with antibodies against PCNA. (C) Colocalization of endog-
enous PCNA foci and GFP in V79 cells transiently transfected with
GFP-Fen1 plasmid and preextracted with CSK buffer containing
0.5% Triton X-100. Bar, 10 ?m.
Evidence of interaction of GFP-Fen1 protein with endog-
Protein Mobility at Replication Foci
Vol. 16, May 20052521
suggests that GFP-CSA can transiently interact with an im-
mobile nuclear structure, e.g., with chromatin. However, we
have found that the redistribution time of GFP-CSA is not
significantly changed after treatment of cells with an inhib-
itor of histone deacetylases, trichostatin A, or UV (Figure
5C). It may be noted that an absence of increased residence
time of GFP-CSA after UV-irradiation suggests that a major
fraction of this protein is not immobilized at TCR foci, but
immobilization of a small fraction of GFP-CSA cannot be
The function of the mobile Pol? associated with normal
replication foci is unknown, but it may be involved in the
bypass of spontaneous single-strand DNA lesions (Avkin
and Livneh, 2002; Kusumoto et al., 2002), which are gener-
ated at about ?1000 per cell per hour (Vilenchik and Knud-
Transient Decrease of Mobility of GFP-Fen1 after
Treatment of Cells with Methyl Methanesulfonate
Some DNA lesions induced by MMS in mammalian cells
cannot be copied by replicative DNA polymerases, leading
to the stalling of replication forks at DNA lesions (Merrick et
al., 2004) and to an accumulation of S-phase cells with Pol?
foci (Kannouche et al., 2001). The treatment of cells with 0.02
and 0.03% MMS (but not with 0.01% MMS) decreases the
rate of movement of replication forks (Merrick et al., 2004),
which has been shown by the replicative labeling of DNA
fibers (Tomilin et al., 1993; Jackson and Pombo, 1998). Using
FRAP assay, we examined whether the mobility of focal
GFP-Fen1 is effected by MMS treatment. Figure 6, A and B,
shows results obtained with stably expressing cells, and it is
seen that there is a change in mobility of focal GFP-Fen1
when cells are analyzed 2–3 and 5–6 h after treatment with
0.03% MMS, but normal mobility is observed 8–9 h after
DNA damage. Because analysis of each cell takes a few
minutes and many cells should be analyzed, we averaged
FRAP curves obtained within the indicated time intervals.
The mobile fraction of focal GFP-Fen1 is strongly decreased
(Figure 6C), and its residence time at replication foci is
increased (Figure 6D) at 2–6 h after 0.03% MMS, but when
analyzed 8–9 h or at 24 h focal GFP-Fen1 showed normal
mobility (Figure 6, C and D). A lower concentration of MMS
(0.01%) also induced a slight decrease of the mobile fraction
of focal GFP-Fen1 at 2–3 h after treatment (Figure 6C), but it
did not effect its residence time. A strong decrease of the
mobile fraction of focal GFP-Fen1 at 2–3 h after treatment
with 0.03% MMS also was observed with transiently trans-
fected V79 cells (Supplementary Figure 3).
The observed changes of mobility of focal GFP-Fen1 after
MMS correlate with the ability of this agent to slowdown the
movement of replication forks (Merrick et al., 2004) consis-
tent with the view that they are induced by the stalling of
replication forks. However, like ionizing radiation (Painter
and Young, 1980; Larner et al., 1999), MMS also suppresses
the initiation of new replicons (Merrick et al., 2004). Here, by
using FRAP assay, we studied the mobility of focal GFP-
Fen1 in transiently transfected Chinese hamster cells after
their treatment with radiomimetic bleomycin which sup-
presses DNA synthesis only through the inhibition of repli-
con initiation (Noda, 1988; Liapunova et al., 1989). We did
not find any changes compared with the control in the
recovery of GFP-Fen1 fluorescence after treatment of cells
with 200 ?g/ml bleomycin (Figure 7A). Bleomycin activity
was confirmed by the analysis of ?-H2AX foci (Supplemen-
tary Figure 4) and of cell survival (Figure 7B). This result is
consistent with the view that the decrease of the mobile
expressing GFP-CSA protein. Left two images show transiently (top) or stably (bottom) transfected living cells, and right two images show
unirradiated (top) or UV-irradiated (bottom) stably transfected cells preextracted with CSK buffer containing 0.5% Triton X-100 (Kamiuchi
et al., 2002). Bar (A), 10 ?m. UV dose was 34 J/m2followed by 1-h incubation of cells in growth medium. (B) Survival of stably transfected
GFP-CSA expressing cells (clone OS-7) after UV, parental cells (CS3BESV), and normal human fibroblasts (line WI38VA13). (C) Immunoblot
of proteins from OS-7 cells probed with antibodies against GFP. (D) UV-induced insolubilization of GFP-CSA protein in OS-7 cells and its
stimulation by pretreatment with trichostatin A (300 nM, 24 h). Roscovitine (20 ?M) was added for 6 h before UV-irradiation. Mean GFP-CSA
fluorescence intensity ?50 of nuclei of OS-7 cells was measured under identical conditions (amplification gain and magnification) for all
Expression of GFP-CSA protein in human cells (CS3BESV) with mutation of endogenous CSA protein. (A) Images of cells
L. Solovjeva et al.
Molecular Biology of the Cell2522
fraction of GFP-Fen1 after MMS is caused by an inhibition of
movement of replication forks and not by an inhibition of
The mobile fraction of focal GFP-Pol? is not significantly
changed after treatment with 0.03% MMS at 2–3, 5–6, and
8–9 h (Figure 8A). The residence time of GFP-Pol? is slightly
decreased at 5–6 h after 0.03% MMS but is not changed at
2–3 h (Figure 8B), when a strong decrease of the mobile
fraction and an increase of resident time of GFP-Fen1 were
observed (Figure 6). The low mobility of GFP-PCNA is not
changed after treatment with 0.03% MMS (Figure 8, C and
D), although we observed significant monoubiquitination of
endogenous PCNA in V79 cells 5 h after lower doses (0.01%)
of MMS (Figure 8E, lane 2). It seems, therefore, that GFP-
Pol? remains very mobile during extensive lesion bypass
after MMS damage, which can be functionally important for
the efficient polymerase switch at stalled replication forks.
Within the nucleus, DNA synthesis is initiated sequentially
at spatially distinct RFi, in which clusters of replication forks
(Jackson and Pombo, 1998) accumulate the specific proteins
required for DNA synthesis. The accumulation of GFP-
tagged variants of these proteins at RFi are seen in living
cells, and analyses of their mobilities by using the FRAP
assay showed that at least one of them, PCNA, is slowly
exchanged (Sporbert et al., 2002). We also found that the
focal GFP-PCNA has a low mobility in normally proliferat-
ing cells, but two PCNA-binding proteins associated with
the RFi in normally proliferating cells, GFP-Fen1 and GFP-
Pol?, are very mobile with residence times at replication foci
?0.8 and 2 s, respectively. The redistribution time of focal
GFP-PCNA is much longer, and it has been shown that some
of the recovery of fluorescence observed in 45 min after
bleaching occurs not exactly at the bleached sites but at
closely adjacent sites (Sporbert et al., 2002). This may be
caused by a slow exchange of PCNA in the moving replica-
tion complex with free PCNA molecules or by de novo
assembly of PCNA clamps within a single large focus
(Sporbert et al., 2002). The lifetime of GFP-PCNA foci in
mid-late S-phase cells can reach 3 h (Leonhardt et al., 2000),
comparable with the lifetime of mammalian replicons in
mid-late S phase, which is ?3.5 h (Liapunova, 1994).
Together, our results support a dynamic model of the
normal replication complex in which relatively long-living
and stable DNA-bound PCNA-based holocomplexes bind
mobile subunits with low residence time comparable with
the time of synthesis of one Okazaki fragment. Our results
also directly support the concept of self-organization in cel-
lular architecture (Misteli, 2001). It will be of interest to
study the mobilities of other replication proteins, but the
existence of at least three mobile components in the replica-
transfected (A and B) or stably expressing cells (C and D) and
GFP-Pol? in transiently transfected cells (E and F). Bar (A, C, and E),
5 ?m; numbers show seconds after photobleaching. In B, D, and F,
vertical bars represent SEs of average values from 10 to 30 cells for
each variant. Bleached segments are boxed.
FRAP analyses of mobility of GFP-Fen1 in transiently
Redistribution times of GFP-Fen1, GFP-Pol?, GFP-PCNA, and GFP-CSA
2 h after 0.03% MMS
24 h after 0.03% MMS
Normalization of the averaged original recovery curves has been done using equation RFtm(t) ? (It? I0)/(Ipost? I0). Value of tm
(representative residence time for focal component) was calculated by approximation from the normalized curves as time required for 63.2%
recovery of the fluorescence intensity after complete recovery as described previously (Lukas et al., 2003). For mathematical reasons, the SD
of tmis always as large as the tmitself (Lukas et al., 2004) and is not shown here.
Protein Mobility at Replication Foci
Vol. 16, May 20052523
tion machinery (RPA34, Fen1, and Pol?) indicate that repli-
cation complexes are not very different from excision repair,
splicing, recombination, and transcription complexes, which
are usually assembled from mobile proteins (Houtsmuller et
al., 1999; McNally et al., 2000; Phair and Misteli, 2000; Dundr
et al., 2002; Essers et al., 2002). High mobility is important for
functional plasticity, because it allows different proteins to
interact with one and the same target and produce different
complexes with common subunits. The very low exchange
of PCNA in replication foci may seem to be exceptional, but
it should be noted that the half recovery time of transcrip-
tion-engaged mammalian RNA polymerase II (Pol II) in the
FRAP assay in normal living cells is ?20 min (Kimura et al.,
2002). Nonengaged free GFP-Pol II is half-recovered in
?0.25 min (Kimura et al., 2002), which is comparable with
the redistribution time of the GFP-CSA in undamaged, UV-
irradiated or trichostatin A-treated OS-7 cells (3–4 s). This
indicates that the majority of GFP-CSA protein is not stably
associated with the transcription-engaged Pol II during nor-
mal or stalled transcription but may be present in the com-
plex formed by nonengaged Pol II. However, the association
with Pol II transcription sites of a minor fraction of CSA
protein after UV irradiation (Kamiuchi et al., 2002; Supple-
mentary Figure 2) cannot be excluded using the FRAP assay.
Using the FRAP assay, we also have found that introduc-
tion of DNA damage by treatment of cells with MMS leads
to a strong but transient decrease of the mobile fraction of
GFP-Fen1. The expression of nuclease-defective Fen-1 in
mammalian cells causes a prolonged delay of S-phase pro-
gression after MMS treatment (Shibata and Nakamura,
2002), indicating involvement of this nuclease in the process-
ing of stalled replication forks. Because Fen1 is known to be
recruited to replication through interactions of its conserved
C-terminal domain with the interdomain connector loop
(IDCL) or C-terminal domain of PCNA (Li et al., 1995; Jon-
sson et al., 1998; Gomes and Burgers, 2000; Tom et al., 2000),
it is possible that DNA damage-induced immobilization of
Fen1 reflects modulation of its interactions with PCNA. It is
Mobile fractions of focal GFP-Fen1 and GFP-Pol? before and after treatment of cells with MMS
MF at different times after MMS treatment (h)a
0 0.52.5 5.58 24
GFP-Fen1 (0.01% MMS)
GFP-Fen1 (0.03% MMS)
GFP-Pol? (0.03% MMS)
0.89 ? 0.02
0.89 ? 0.02
1.03 ? 0.04
1.00 ? 0.02
0.89 ? 0.04
0.82 ? 0.03
0.78 ? 0.06
0.40 ? 0.04
0.92 ? 0.03
0.86 ? 0.01
0.42 ? 0.04
0.88 ? 0.04
0.90 ? 0.02
0.89 ? 0.04
0.93 ? 0.02
0.97 ? 0.03
0.94 ? 0.04
0.95 ? 0.03
aMF is calculated as maximal value of the function RFmf? (It–I0)/(Ipre–I0), where Ipreis the initial fluorescence before photobleaching (taken
as 1). First row (0 time) shows values obtained for the control cells not treated with MMS.
fected V79 (A) or stably expressing OS-7 cells (B and C). Vertical
bars in A show SE; numbers in B show seconds after photobleach-
ing. Bar (B), 10 ?m; bleached segment is boxed. Redistribution times
in C were calculated from the original FRAP curves as mean times
for recovery of 50–75% of the maximal fluorescence recovery ? SD.
Trichostatin A (TrA; 300 nM) was added for 24 h, UV-dose was 34
J/m2followed by 1-h incubation in growth medium.
FRAP assay of mobility of GFP-CSA in transiently trans-
L. Solovjeva et al.
Molecular Biology of the Cell 2524
unlikely that Fen1 immobilization is a consequence of the
binding to the IDCL of PCNA of the cell cycle inhibitor
p21/Cip1 (Zhang et al., 1998), because p21 inhibits Fen1
binding to PCNA (Jonsson et al., 1998). It is also unlikely that
MMS-induced immobilization of Fen1 is caused by its phos-
phorylation at Ser-187 by Cdk1/cyclin A and Cdk2/cyclin A
complexes, which also inhibits its binding to PCNA (Hen-
neke et al., 2003). However, we cannot exclude that the Fen1
immobilization is a consequence of DNA damage-induced
hyperacetylation of Fen1 protein (Hasan et al., 2001), or of
Rad18-dependent monoubiquitination of PCNA (Hoege et
al., 2002; Kannouche et al., 2004; Watanabe et al., 2004).
Stalling of DNA polymerase ? at DNA lesions may change
contacts of this polymerase with the IDCL of PCNA (Jonsson
et al., 1998; Zhang et al., 1998; Ducoux et al., 2001; Lu et al.,
2002). Pol? also interacts with PCNA through the conserved
domain at the Pol? C terminus (Haracska et al., 2001; Kan-
nouche et al., 2001), which is very similar to that of Fen1, but
the Pol? mobile fraction and residence time are not signifi-
cantly changed after an MMS dose, which induces strong
immobilization of GFP-Fen1. This indicates that it is not
modulation of Fen1 binding to PCNA but some other factors
that can effect Fen1 mobility after DNA damage. If Fen1
detachment from the PCNA clamp during normal lagging
strand synthesis is possible only after cutting of the single-
stranded tail displaced by Pol?/PCNA complex from the
previous Okazaki fragment (Waga and Stillman, 1998; Maga
et al., 2001), Fen1 can stay associated with PCNA upon
blocking of Pol?/PCNA at the lesion in the template strand
and restore its mobility only after elimination of the block. In
this scenario, stalling of replication forks at lesions is a direct
cause of decreased Fen1 mobile fraction.
MMS-induced changes of GFP-Fen1 mobility found in this
study also may be interpreted as an indication of the in-
volvement of this protein in some aspects of lesion bypass. It
is known that in yeast the Rad27 gene, which belongs to the
Rad6 epistasis group (Reagan et al., 1995), suppresses the
accumulation of ssDNA and DSBs at replication forks (De-
brauwere et al., 2001). In mammalian cells, nuclease activity
of Fen1 promotes S-phase progression after MMS damage
(Shibata and Nakamura, 2002), and this activity of Fen1 at
replication foci in MMS-treated cells may be directly re-
quired for efficient processing of single-strand gaps after
DNA polymerase switch and translesion synthesis. This pro-
cessing may take more time than the elimination of 5? flaps
during normal replication explaining the transient increase
of the residence time of mobile fraction of focal GFP-Fen1 in
MMS-treated cells (Figure 6D). An increased residence time
of Fen1 at replication foci already detectable 30 min after
ity of GFP-Fen1 in stably expressing V79
cells. In A, bleached segment is boxed; bar is
5 ?m, and numbers show seconds after
bleaching. Vertical bars in B show SE of
average values for 10–30 cells, results ob-
tained at 2–3, 5–6, and 8–9 h after MMS
treatment are combined. (C) Mobile frac-
tions of GFP-Fen1 (circles) or GFP-Pol? (tri-
angles) at different times after MMS calcu-
lated as described in Materials and Methods.
(D) Changes of residence time of GFP-Fen1
at replication foci after MMS, calculated as
described in Materials and Methods.
MMS-induced changes of mobil-
transfected V79 cells with bleomycin (A) and survival of V79 cells
after bleomycin (B). Treatment with bleomycin in B was for 2 h.
Vertical bars in A show SE from 10 cells; vertical bars in B show SD
from three experiments.
Mobility of GFP-Fen1 after treatment of transiently
Protein Mobility at Replication Foci
Vol. 16, May 20052525
MMS treatment (Figure 6D) may represent the dynamic
signal for activation of DNA damage checkpoints required
for accumulation of mammalian Rad18 at stalled replication
foci (Nikiforov et al., 2004) and Rad18-dependent mono-
ubiquitination of PCNA (Kannouche et al., 2004; Watanabe et
al., 2004). Further studies are clearly required to establish
actual functional significance of dynamic changes of Fen1 at
stalled replication forks.
We are grateful to P. C. Hanawalt for support in performing some experi-
ments at the Confocal microscopy facility of the Stanford University, A. R.
Lehmann for plasmid encoding GFP-Pol? and for helpful discussions; and V.
Tomilin for help in the treatment of FRAP curves. This research was sup-
ported by the Office of Science (Biological and Environmental Research); U.S.
Department of Energy grant DE-FG03-01ER63070; the Russian Fund for Basic
Research grants 02-04-49145 and 04-04-49292; the Russian Federal State Con-
tract 10002-251/P-10/143-173/010403-049; and the Russian Academy of Sci-
ences program MCB.
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